I intend to isolate, purify and characterize the LDL (low density lipoproteins) receptors present on the membrane of cultured human skin fibroblasts. I have available normal skin fibroblasts, fibroblasts from patients who have abnormal or missing receptors for LDL or have an abnormal intracellular and pericellular accumulation of glycosaminoglycans. Normal or mutant fibroblasts are grown in plastic bottles (using defined media supplemented with complete or lipoprotein-free fetal calf serum) and labeled with 3H-leucine or 3H-glucosamine and 35SO4 for a period of 48 hour. After removal of the medium, the cells will be treated sequentially with collagenase and trypsin. Cells from similar cultures, not labeled, will be scraped into 0.05 M Tris-HCl buffer pH 7.4 (O.15 M NaCl), then frozen in liquid nitrogen for the assay of the enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase, EC 1.1.1.34). The medium, the collagenase and the trypsin pools will be applied to separate affinity columns in which LDL has been covalently linked to CNBr-activated Sepharose B. Each column will be eluted with saline, 0.5 M KCl, 2.5 M KCl and 2.5 M KI. Each eluent, properly diluted, is assayed for hemagglutinating activity using LDL-coated formocells. Material possessing agglutinating activity will be dialyzed, assayed for radioactivity, purified and analyzed. The receptors will be throughly analyzed to discern whether they are proteins, glycoproteins, glycosaminoglycans or proteoglycans. On a second series of experiments, cell membranes will be isolated by density gradient centrifugation and then extracted with 6 M guanidinum chloride, in order to obtain the receptors without use of proteases. The receptors will be isolated with similar techniques from mutant fibroblasts; their affinity for LDL will be compared to that of wild-type fibroblasts, using techniques of hemagglutination inhibition. With this approach, I should be able to detect functional differences between normal and abnormal receptors.